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1398
Jing
et al.
: J
ournal of
AOAC I
nternational
Vol. 98, No. 5, 2015
when stored in a refrigerated (8°C) autosampler. Filtered
samples (aqueous) and WS are stable for 7 days stored at 2–8°C.
(
d
) The method can also determine free choline and free
carnitine by bypassing the microwave digestion and basic
hydrolysis. Simply dilute the same sample size with water, add
125 µL of MIX-IS, and dilute to 10 mL in a tube. Mix, and then
dilute this solution 1 mL to 25 mL with water in another tube.
Mix, filter approximately 1 mL of sample slurry, and then dilute
10x with acetonitrile as above. No nitric acid or ammonia is
used.
G. Instrument Operating Conditions
(
a
)
MS conditions.—See
Tables
2014.04D
and
E
.
(
b
)
UPLC conditions.
–
See
Tables
2014.04F
and
G
.
(
c
)
UPLC analysis
.—Column stability was improved by
storing the column in water–acetonitrile (5 + 95, v/v) without
Table 2014.04E. Mass spectrometer (Xevo TQ-S)
operating conditions
Ionization mode
ESI positive
Capillary voltage, kV
2.0
Collision gas pressure, mtorr
2–5 × 10
–3
Source temperature,
°
C
150
Source offset, V
30
Desolvation temperature,
°
C
550
Cone gas flow, L/h
300
Desolvation flow rate, L/h
1000
Peak width half-height, amu
a
0.7
a
Instrumental resolution parameters for the TQ-S are set up by
IntelliStart to achieve a resolution of approximately 0.7 amu across
the mass range.
Table 2014.04F. Chromatographic parameters
Mobile phase A
10 mM ammonium acetate
[water–acetonitrile (95 + 5, v/v)]
Mobile phase B
10 mM ammonium acetate
[water–acetonitrile (5 + 95, v/v)]
Flow rate
0.7 mL/min (analytical)
Flow rate into MS
Full flow
Column
Acquity UPLC BEH HILIC
1.7 µm, 2.1 × 100 mm
Column temperature,
25
Injection volume, µL
10
Injection type
Full loop
Sample temperature,
°
C
8
Table 2014.04G. Gradient profile
Mobile phase
Time, min Flow, mL/min
A, %
B, % Curve
Initial
0.7
8.0
92.0
0.10
0.7
8.0
92.0
6
6.00
0.7
22
78
6
6.01
0.7
100
0
6
8.00
0.7
100
0
6
10.00
1.0
8.0
92.0
6
13.00
1.0
8.0
92.0
6
13.20
0.7
8.0
92.0
6
15.00
0.7
8.0
92.0
6
Table 1. Method performance requirements
Choline (a)
Carnitine (b)
Analytical range
2–250
0.16–20
LOD
0.7
NA
a
LOQ
2.0
≤0.16
Repeatability (RSD
r
)
10% at 2 mg/100 g;
otherwise 5%
≤8%
Recovery
90–110% at all levels
over range
90–110% at all levels
over range
Reproducibility (RSD
R
)
15% at 2 mg/100 g;
otherwise 10%
≤15%
(a) mg/100 g concen-
trations apply to: (
1
)
“ready-to-feed” liquids
“as is”; (
2
) reconsti-
tuted powders (25 g
into 200 g water); and
(
3
) liquid concentrates
diluted 1:1 by weight.
For all concentra-
tions, choline will be
expressed as mg/100 g
reconstituted liquids.
Report as total choline.
(b) mg/100 g concen-
trations apply to: (
1
)
“ready-to-feed” liquids
“as is”; (
2
) reconsti-
tuted powders (25 g
into 200 g water); and
(3) liquid concentrates
diluted 1:1 by weight.
a
NA = Not applicable.
Table 2. SLV test materials
Product type
Code
Sample size used
for repeatability
SLV, g
SRM 1849a
NA
c
5.0
a
Infant powder milk
D04HTCVV
4.3
a
Infant powder soy
E29JVLV
3.7
a
Infant powder milk part hydrolyzed 1172572116
3.9
a
Infant powder soy part hydrolyzed 117257651Z
4.1
a
Adult powder low fat
00394RF00
4.1
a
Adult powder milk
11750017V3
7.5
a,b
Child formula powder
00412RF00
2.7
a
Infant elemental powder
00403RF00
3.7
a
Infant RTF milk
SPIFAN CTL
3.0
Adult RTF high protein
00414RF00
0.8
Adult RTF high fat
00406RF00
0.6
a
Aliquot size from an 11.1% (w/w) reconstitution in water (25 g powder
plus 200 g water).
b
An abnormally large sample size was needed for this sample, in which
both choline and carnitine were present at <LOQ.
c
NA = Not applicable.
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